Although the field of genomics has recently sequenced thousands of genes from multiple species, it is much slower at providing information on assigning functions to these genes. Proteins are generally the effector molecules that ascribe roles to genes, and most of the functions of proteins arise through their three-dimensional structures and their interactions with other molecules. Conventional genetic and structural biology techniques have, respectively, been the most powerful avenues for determining the organization of cell signaling networks and the molecular details of protein structure and protein/protein interactions. Understanding protein structure and the protein:protein interactions of the cell at a basic level (i.e. identification of the molecules involved, determination of their molecular architecture, and elucidation of how these molecules interact with one another) is imperative for understanding how the disruption of a single element can result in human disease. X-ray crystallography and nuclear magnetic resonance spectroscopy are the current methods of choice for obtaining high resolution structural information. Mass spectrometry (MS) is an emerging technique that is showing tremendous potential for both identification of protein complexes and elucidation of protein structure, in particular for proteins that are not amenable to classical structural techniques. The advantages of MS sensitivity, low sample consumption, and the ability to analyze inhomogeneous mixtures can overcome the obstacles that hamper other structural methods. MS was actually developed about 100 years ago, but its utility in biological research is just now being realized. Using MS, we can probe the structure of a protein with chemical reagents and then assess inter-residue distances and solvent accessibility. These data can aid in the determination of the protein structure and, hence, as to how the protein works. Moreover, MS can also be employed to simultaneously determine what other proteins a particular protein of interest interacts with and how these interactions are formed. Protein:protein and protein:DNA interactions are often at the center of biological processes, both beneficial and harmful. The primary goal of this project is to determine structural features of protein interactions that are critical in biologically functional or in pathological processes. As examples, we are currently studying DNA repair enzyme interactions with DNA, the interaction surfaces in DNA mismatch repair enzymes, and structural transformations of proteins concomitant with phosphorylation that are involved in carcinogenesis. MLH1:PMS1 heterodimer: DNA mismatch repair (MMR) is critical for the maintenance of the genetic material, and the major features have been conserved throughout evolution. MutL proteins are primary components of MMR with MutL homolog 1 (MLH1) and Post meiotic segregation 1 (PMS1) critical components in yeast. These proteins form a heterodimer via the C-terminal domain. Upon DNA and ATP binding, further dimer contacts are thought to form in the N-termini. Little is known about their tertiary structure. It is hypothesized that mutations in these proteins cause changes in their structures, which may lead to loss of MMR activity and ultimately carcinogenesis. The 3-D structures of yeast MLH1 and PMS1 are being probed through the use of differential acetylation. The complex, itself, is currently being expressed. Specific aims that have been achieved:Model the structure of yeast MLH1 based on homology modeling and determination of surface accessible residues; Determine contact and binding sites of MLH1 and PMS1 heterodimer by differential surface modification of the heterodimer and monomeric MLH1; Map DNA binding sites in yeast PMS1 NTD using limited proteolysis. Hepatitus C mAb: Hepatitis C virus infects over 170 million people worldwide, and in most cases, the infections develop into chronic hepatitis, which is one of the most prevalent causes of liver cirrhosis and represents the most frequent indication for liver transplantation. Hepatitis C virus (HCV) is a small, enveloped positive-strand RNA virus belonging to the Flaviviridae family. The genome of HCV is ~ 9500 nucleotides and contains a single large open reading frame encoding a single polypeptide of ~3010aa. This polyprotein is subsequently processed co-and posttranslationally, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. HCV-E2 is a 50kDa glycoprotein that shows large variations among HCV genotypes and contains a hypervariable 27 aa sequence at its amino terminus. Specific aims: 1.To characterize the structure of the HCV-E2 protein and of the E1E2 protein complex by peptide mapping using mass spectrometry; 2.Determine epitopes of the E2 protein, recognized by in vivo neutralizing, human mAbs. Status:a)Peptide mapping is being performed on HCV-E2 protein and the E1E2 protein complex and have partially confirmed the sequences of the proteins; b)Initial experiments have been performed on 3 neutralizing mAbs CBH2, CBH5 and CBH7. MS/MS experiments are being performed to characterize the structure of the epitope fragments. Mapping sites of interaction of Sindbis coat glycoprotein in the intact virus: We are using surface modification to identify surface E2 glycoprotein, and on the E1 glycoprotein, all in agreement with previously-published structural data. We are now repeating the experiment using virus that has been transiently exposed to acidic pH. This induces a permanent conformational change. Comparison of results between the 2 experiments will help understand the conformational change that occurs. Temperature-dependent oxidation kinetics as a measure of protein dynamics: We have shown using apomyoglobin that the dynamic solvent interfaces in proteins can be detected by examining the temperature dependence of the oxidation kinetics of side chains. Dynamic solvent interfaces show temperature-dependent oxidation kinetics, while stable solvent interfaces do not. Further experiments on 2 proteins with known dynamic regions, SUMO and Spo0F are under investigation.